Column Overloading?

[Ricardo]

I am doing organic acid analysis and am seeing some changes in retention times for my external stdandard mix. The components are eluting about 0.1 mimutes sooner than previously. I am injecting anywhere from 400 to 11, 000 ppm, as that is what range my samples are in. I also am getting very good repeatability with the std but not with realtime samples. I am using polypropylene inserts, as I have only about 0.2 mls of each sample. Should I be diluting? Please help. My parameters are: Agilent Stablebond AQ column at 1.0 mls/min, at 30 C, 210 nm on the DAD, 20 mM pH 2.9 phosphate buffer, with 1% ACN. Injection volumes for my calibration are 1, 2, 3, 4, and 5 ul.

[tom jupille]

10,000 ppm = 10 ug/uL.

If I read your post correctly, that means that you are getting something over 50 micrograms at the high end (5 uL @ 11 ug/uL). That is definitely pushing things (you didn't say, but I'll assume a 150 x 4.6-mm column).

Are you seeing a variation in retention time with increasing concentration? And are you seeing increased tailing at increasing concentration? Both of those would point toward overload. Also, depends on retention time. A 1% shift from run to run might not be significant.

[Ricardo]

Yes, those are the column dimensions.The shift in retention times is minimal, about 1 %. There is a small amount of peak tailing. I have reduced the concentration of my standard by factor of 2 for the components that have the strongest signal and am rerunning it now.

Thanks for your advice.

Ricardo